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Ivyspring International Publisher Theranostics 2017; 7(13): 3293-3305. doi: 10.7150/thno.19988 Research Paper EGLN1/c-Myc Induced Lymphoid-Specific Helicase Inhibits Ferroptosis through Lipid Metabolic Expression Changes Yiqun Jiang1, 2, 3, Chao Mao1, 2, Rui Yang1, 2, Bin Yan1, 2, 4, Ying Shi1, 2, Xiaoli Liu1, 2, Weiwei Lai1, 2, Yating Liu1, 2, Xiang Wang3, Desheng Xiao5, Hu Zhou6, Yan Cheng7, Fenglei Yu3, Ya Cao1, 2, Shuang Liu4, Qin Yan8, Yongguang Tao1, 2, 3, 4

1. Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008 China; 2. Cancer Research Institute, Central South University, 110 Xiangya Road, Changsha, Hunan, 410078 China; 3. Department of Thoracic Surgery, Second Xiangya Hospital, Central South University, Changsha, China; 4. Institute of Medical Sciences, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008 China; 5. Department of Pathology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008 China; 6. Shanghai Institute of Material Medica, Chinese Academy of Sciences (CAS), 555 Zu Chongzhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, China; 7. Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410078 China; 8. Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 U.S.A.

 Corresponding author: Y.T. Email: [email protected]; Tel. +(86) 731-84805448; Fax. +(86) 731-84470589.

© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2017.03.08; Accepted: 2017.06.15; Published: 2017.07.23

Abstract Ferroptosis is a newly discovered form of non-apoptotic cell death in multiple human diseases. However, the epigenetic mechanisms underlying ferroptosis remain poorly defined. First, we demonstrated that lymphoid-specific helicase (LSH), which is a DNA methylation modifier, interacted with WDR76 to inhibit ferroptosis by activating lipid metabolism-associated , including GLUT1, and ferroptosis related genes SCD1 and FADS2, in turn, involved in the Warburg effect. WDR76 targeted these genes expression in dependent manner of LSH and chromatin modification in DNA methylation and histone modification. These effects were dependent on iron and lipid reactive oxygen species. We further demonstrated that EGLN1 and c-Myc directly activated the expression of LSH by inhibiting HIF-1α. Finally, we demonstrated that LSH functioned as an oncogene in lung cancer in vitro and in vivo. Therefore, our study elucidates the molecular basis of the c-Myc/EGLN1-mediated induction of LSH expression that inhibits ferroptosis, which can be exploited for the development of therapeutic strategies targeting ferroptosis for the treatment of cancer.

Key words: Ferroptosis; LSH; WDR76; EGLN1; PHD2; c-Myc; HIF-1α; SCD1; FADS2; Iron; Lipid reactive oxygen species; Metabolism; Lung cancer.

Introduction Reprogramming of cellular metabolism, iron carrier protein transferrin, and other related including the regulation of apoptotic and necrotic cell regulators, such as glutathione peroxidase 4 (GPX4) death, is necessary for tumorigenesis [1-3]. and p53 [4-7]. Iron and iron derivatives are essential Ferroptosis, which is a newly discovered mode of for the functioning of ROS-producing , non-apoptotic cell death, involves a metabolic suggesting that iron might function as a trigger or dysfunction that results in the intracellular metabolic mediator of cell death signaling, particularly in process glutaminolysis and the production of ferroptosis [4, 8], further indicating that iron-dependent reactive oxygen species (ROS), the iron-containing enzymes are essential for ferroptosis.

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The iron-, oxygen-, and 2-oxoglutarate-dependent Although cancer epigenetic discoveries over the past dioxygenase family includes three members of prolyl decade have revealed numerous epigenetic modifiers hydroxylase domain (PHD) enzymes (PHD 1, 2 that are involved in the progression of various cancers and–3), which are also known as EGLN-2, -1 and -3 [28], the role of chromatin remodeling in lung cancer respectively. EGLN2 is essential for cell death as a remains unclear. candidate driver of iron chelation-mediated inhibition In this study, we investigated the potential of cell death [9]. Investigations are required to impact of LSH on ferroptosis and lung cancer. We determine whether and how the iron-dependent found that LSH is an oncogene and regulates hydroxylase activity of EGLNs contributes to ferroptosis in lung cancer. Mechanistically, LSH ferroptosis. epigenetically increases the expression level of The EGLNs catalyze hypoxia inducible metabolic genes that decrease ferroptosis. Moreover, transcriptional factor (HIF) prolyl hydroxylation, EGLN1 is essential for the induction of the expression leading to the degradation of the oxygen sensors of LSH by counteracting the recruitment of HIF-1α HIF‐1α and HIF‐2α that are necessary for the function rather than that of c-Myc. of the HIFs, whereas HIF-1 regulates the oxygen-dependent metabolism of glucose and Material and Methods glutamine [10, 11]. HIF-1 regulates oxygen-dependent Cell culture, antibodies, chemicals, plasmids glucose, glutamine metabolism and EGLN activity and siRNAs [12-14]. Interestingly, lactate, a common product of anaerobic metabolism, promotes a HIF-dependent The normal lung cell lines MRC-5 (ATCC: hypoxic response, which subsequently triggers CCL-171™) and HBE (ATCC: CRL-2741™) were signals for cell growth and angiogenesis [15]. purchased from ATCC. The lung cancer cell lines Mitochondrial ROS inhibit ELGNs to induce HIF [12]. A549 (ATCC: CCL-185™), H358 (ATCC: CRL-5807™), The relationship between ferroptosis-associated lipid and H522 (ATCC: CRL-5810™) were obtained from ROS production and EGLN activity remains unclear. ATCC. The lung cancer cell lines PC9, 95C and 95D The function of HIF in tumors is well characterized, were obtained from the Cancer Research Institute of but our understanding of the function of EGLNs in Central South University. The A549 cells were tumors is limited. maintained in DMEM/F12 1:1(Hyclone); 293T cells Chromatin-modifying enzymes utilize key were maintained in DMEM (Gibco); other cells were components of core metabolic pathways as co-factors maintained in RPMI 1640 (Gibco). All media were and substrates for signal integration and nuclear supplemented with 10% (V/V) FBS. All cell lines were adaption to environmental changes [16-18]; however, maintained at 37°C with 5% CO2. The cell lines were the mechanisms by which the signal transduction negative for mycoplasma contamination. All cell lines pathways directly communicate with chromatin to were passaged less than 10 times after their initial change the epigenetic landscape are poorly revival from frozen stocks. All cell lines were understood. Lymphoid-specific helicase (LSH), which authenticated by short tandem repeat profiling prior is a protein that belongs to the SNF2 family of to use. chromatin-remodeling ATPases, is critical for the The primary antibodies for LSH and WDR76 normal development of plants and mammals because were purchased from Santa Cruz, the primary it establishes correct DNA methylation levels and antibodies for EGLN1, EGLN3 and HIF1α were patterns [19-22]. LSH maintains genome stability in obtained from Novus (Littleton, CO), the primary mammalian somatic cells [23, 24]; however, the role of antibodies for SCD1 and FADS2 were purchased from LSH in ferroptosis remains unknown. Origen (Rockville, MD); the primary antibody for Lung cancer is a leading cause of death β-acting was purchased from Sigma-Aldrich (St. worldwide and is the first leading cause of death in Louis, MO). The chemicals (erastin, ferrostatin-1, China, resulting in more than 2.8 million deaths in DMOG and BAY 85-3934) were purchased from 2015, more than 40% of which involved lung Selleck (Houston, TX). adenocarcinomas [25]. Furthermore, lung cancer can The LSH lentiviral construct was generated by be divided into small cell lung cancer and non-small inserting the LSH cDNA into plvx-EF1a-puro vector cell lung cancer (NSCLC), including adenocarcinoma (Clontech, Mountain View, CA). (ADC) and squamous cell carcinoma (SCC), which FLAG-EglN1-pLenti6 was a gift from William Kaelin account for 80% to 85% of all lung cancer cases.[26]. (Addgene plasmid # 36949). The Lentiviral shRNA Epigenetic modifications, including chromatin clones targeting human LSH, FADS2, SCD1, and modifications, play important roles in the EGLN1 and the non-targeting control construct were development and progression of NSCLC [26, 27]. purchased from Genechem (www.genechem.com.cn)

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(Shanghai, China). (Automatic Biochemical Analyzer, 7170A; HITACHI) at the Clinical Biochemical Laboratory of Xiangya Western blot analysis and Co- Hospital (Changsha, China). Immunoprecipitation (Co-IP) assay Details regarding the Western blot analysis and Nude mice and study approval Co- Immunoprecipitation (Co-IP) assay have been The xenograft tumor formation was generally previously described [29], and the detailed procedure performed as previously described [30]. The SCID is listed into the supplementary Materials and Mice were purchased from the Hunan SJA Laboratory Methods. Animal Co., Ltd. (Changsha, China). The procedural details are listed in the supplementary Materials and Cell proliferation assays, migration and Methods. invasion assays and plate-colony formation assays, quantitative real-time PCR and RNA Statistical analyses sequencing All experiments, except for those that involved The details of these procedures have been the nude mice, were repeated at least three times. The previously described [29, 30], and the detailed results are shown as mean ± SD or SEM. All statistical procedure of the RNA sequencing is listed in the analyses were performed using the Prism 6.0 supplementary Materials and Methods. The primer GraphPad software. A p value of less than 0.05 was sequences used are listed in the supplementary Table considered to be statistically significant. S1. The mean ± SD of three independent experiments is shown. Results Immunohistochemical (IHC) analysis of lung LSH activates metabolic gene expression at cancer biopsies, and immunofluorescence the transcriptional level assay We first assessed the expression of LSH in a The IHC analyses were generally performed as panel of lung cells using a Western blot analysis previously described [29, 30]. The lung cancer (Figure 1A) and found an increased expression of LSH biopsies, which were validated by a pathologist (Dr. in the lung cancer cell lines compared to that in the Desheng Xiao of Xiangya Hospital), were obtained normal lung cells in which LSH was expressed at a from the Department of Pathology at Xiangya low level; then, we selected three lung cancer cell Hospital. The lung cancer tissue array was purchased lines, i.e., A549, H358 and PC9, for the subsequent from Pantomics (Richmond, CA, USA). studies. To gain insights into the LSH function, we used RNA-sequencing to analyze gene expression Measurement of total ROS, Lipid ROS, and changes in the cells by comparing the stable intracellular iron expression of LSH to PC9 lung cells transfected with a The details of the procedures have been control vector (Supplementary Figure S1A). We previously described [4, 8]. The detailed procedure is obtained an average of 8.3 million unique mapped listed in the supplementary Materials and Methods. reads and 16995 unique transcripts per condition from two independent biological replicates (fragments per Chromatin immunoprecipitation (ChIP) assay kilobase of exon per million reads (FPKM) >1 in both and hydroxymethylated DNA replicates). After processing the data and averaging Immunoprecipitation (hMeDIP) assay the replicates, compared to the control cells, we The ChIP and hMeDIP assays were performed identified 855 mRNAs that were increased by as previously described [29, 30]. The ChIP DNA was two-fold (upregulated) and 858 mRNAs that were analyzed by qPCR using SYBR Green (Bio-Rad) on an decreased by two-fold (down-regulated) in the LSH ABI-7500 (Applied Biosystems) using the primers expression group (Figure 1B). A volcano plot further listed in supplementary Table S2. The primers were showed 305 upregulated and 423 down-regulated used to amplify in the promoter regions of selected genes (Supplementary Figure S1B). Moreover, the genes. The antibodies were used as indicated. analysis identified a significant enrichment in pathways related to metabolic Glucose uptake and lactate production processes (Figure 1C). measurement Because LSH is potentially linked to metabolic Cells (5×105) were seeded into 6-well plates and genes, we selected two gene subtypes and assessed after incubated for 4 h. The medium was discarded, their expression. The first group comprised glucose and the cells were incubated in fresh medium for 8 h. transporters (GLUTs), which were important in The glucose and lactate levels were measured glucose transport, and the other group comprised

http://www.thno.org Theranostics 2017, Vol. 7, Issue 13 3296 (FADSs), which were dependent increased the expression levels of FADS2 (fatty acid on NAPDH [31]. To substantiate our findings, we desaturase 2) and FADS5 (also known as sterol-CoA stably overexpressed LSH in the H358 lung cancer cell desaturase 1, SCD1). Conversely, we found that the lines (H358-LSH) (Supplementary Figure S1C). depletion of LSH decreased the expression of FADS2, Consistent with the data from PC9 cells, we found SCD1 and FADS6 (Figure 1F-G). Furthermore, we that overexpression of LSH significantly increased confirmed that LSH promoted glucose consumption GLUT1, GLUT6, GLUT12 and GLUT13 expression and lactate production that was associated with the (Figure 1D). To validate the role of LSH in the Warburg effect (Figure 1H-I), whereas depletion of metabolic gene expressions, we stably knocked down LSH decreased glucose consumption and lactate LSH in A549 cancer cells. The knockdown approach production (Figure 1J-K). These results are consistent successfully reduced the LSH protein to less than 10% with a function role for LSH in these processes and the (Supplementary Figure S1D), and the knockdown of observed phenotype. Taken together, LSH affects the LSH decreased the GLUT1, GLUT3, GLUT6, GLUT8 metabolic gene expression as well as the Warburg and GLUT14 expression levels (Figure 1E). Finally, we effect. found that the LSH overexpression significantly

Figure 1. LSH affects metabolic gene expressions at the transcriptional level. (A) A Western blot analysis was used to detect the expression of LSH in a panel of lung cancer cells. (B) LSH induced the down-regulation of 858 genes and the upregulation of 855 genes in the H358 cells. (C) The genes that were positively correlated with LSH in the H358 cells were significantly involved in the regulation of cellular processes and metabolic processes according to the gene ontology analysis using DAVID. (D-G) RT-qPCR analysis of GLUT family associated genes (D, E) and FADS family associated genes (F, G) in LSH-overexpressing H358 cells (D, F) and LSH depletion in A549 cells (E, G). (H-K) Glucose (H, J) and lactate (I, K) levels were determined in the culture medium in H358 and PC9 with LSH overexpression (H, I), and in A549 cells with LSH depletion (J, K). * P<0.05, ** P<0.01, *** P<0.001.

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Figure 2. LSH recruits WDR76 to directly regulate metabolic genes. (A) A549 and H358 cells were assessed to determine the co-localization of LSH and WDR76. (B, C) Co-immunoprecipitation of LSH and WDR76 in A549 cell lysates with anti-LSH antibody (B), and with anti-WDR76 antibody (C) in A549 and H522 cells. (D) ChIP analysis with anti-WDR76 indicated that WDR76 recruitment to the promoters of GLUT1, SCD1 and FADS2 antibodies. (E) ChIP analysis with anti-Flag M2 agarose revealed LSH recruitment to the promoters of GLUT1, SCD1 and FADS2. (F) Re-ChIP assays of Flag-LSH and WDR76 showed WDR76 binding to the promoters of GLUT1, SCD1 and FADS2. (G) ChIP analysis with anti-WDR76 indicated that WDR76 enrichment decreased in the indicated promoters after depletion of LSH. * P<0.05, ** P<0.01, *** P<0.001.

overexpression of LSH promoted the enrichment of WDR76 increases metabolic gene expression WDR76 to the indicated promoters (Figure 2D). through epigenetic regulation in dependent of Similarly, LSH was also directly enriched to the LSH indicated TSSs (Figure 2E). Furthermore, using WDR76 is a nuclear WD40 protein, whose Re-ChIP assay, we further found that WDR76 was function is poorly characterized in mammals. recruited to the promoters of GLUT1, SCD1 and However, WDR40 always functions an active marker FADS2 and that LSH promoted the enrichment of for histone modification (i.e. H3K4Me3). Interestingly, WDR76 at the TSSs of GLUT1, SCD1 and FADS2 our immunofluorescence analysis revealed that (Figure 2F). Finally, the enrichment of WDR76 to the WDR76 co-localized with LSH in A549 and H522 cells indicated genes decreased after depletion of LSH in in the nuclei (Figure 2A). These observations suggest A549 cells (Figure 2G), indicating that WDR76 binds that WDR76 may directly interact with LSH in human to metabolic gene promoter in dependent of LSH. cells. Then, we confirmed the interaction of Our histone modification analysis in the endogenous LSH and WDR76 by Co-IP assay with presence of LSH showed that LSH slightly changed anti-LSH antibodies in A549 and H522 cells (Figure global levels on different histone modifications, 2B). We further confirmed the interaction of including H3K4Me3, H3K27Me3 and H3K9Me3 endogenous LSH and WDR76 by (Supplementary Figure S2B-D). Moreover, the active co-immunoprecipitation assay with anti-LSH and chromatin marker, H3K4Me3, was significantly anti-WDR76 antibodies in A549 and H522 cells higher at the GLUT1, SCD1 and FADS2 promoters in (Figure 2C). the presence of LSH (Supplementary Figure S3A), Because LSH is localized in the nucleus whereas the repressive chromatin marker, and acts as a chromatin modifier dependently and H3K27Me3, decreased at the GLUT1, SCD1 and independently of DNA methylation [32, 33], we FADS2 promoters (Supplementary Figure S3B) upon performed chromatin immunoprecipitation (ChIP) LSH1 overexpression. LSH is a reader of 5-(hydroxyl) assays to determine whether LSH could directly bind methylcytosine (5-hmC) in mice [34]. Furthermore, to the promoters of GLUT1, SCD1 and FADS2 hMeDIP showed that LSH increased the 5-hmC levels (Supplementary Figure S2A). We found that WDR76 at the GLUT1, SCD1 and FADS2 promoters in H358 was directly associated with the indicated cells (Supplementary Figure S3C) and PC9 cells transcription start sites (TSSs) whereas

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(Supplementary Figure S3D). Finally, a Kaplan-Meier depletion of LSH increased the erastin-induced plotter that was performed on a cohort of lung cancers growth inhibition in the A549 cells (Figure 3C), showed that lower WDR76 expression was associated suggesting that LSH might inhibit erastin-induced with overall survival in all lung cancers cancer cell death. The intracellular concentrations of (Supplementary Figure S3E). Similar findings were iron and lipid ROS are both surrogate markers for observed for GLUT1, SCD1 and FADS2, where lower ferroptosis [4, 8]. We found that LSH decreased the expression was associated with overall survival in all intracellular concentrations of iron in H358 and PC9 lung cancers (Supplementary Figure S3E). cells (Figure 3D-E), but LSH depletion increased the intracellular concentrations of iron in the A549 cells LSH inhibits ferroptosis by decreasing the (Figure 3F-G). intracellular levels of iron and lipid ROS Furthermore, we found that lipid ROS levels The LSH effects on the metabolic genes led us to increased after the erastin treatment, and LSH characterize the role of LSH in the novel ferroptotic decreased the erastin-induced lipid ROS in the H358 mode of non-apoptotic cell death, which is associated cells (Figure 3H) and PC9 cells (Figure 3I). The with metabolic dysfunction, by assessing depletion of LSH increased lipid ROS levels in the erastin-induced growth inhibition, in the presence A549 cells (Figure 3J). Moreover, we found that total and absence of ferrostatin, an inhibitor of erastin. ROS were decreased in the presence of LSH in H358 First, we demonstrated that erastin-induced cell death cells (Supplementary Figure S5A-B) and PC9 cells was dose-dependent (Supplementary Figure S4A), (Supplementary Figure S5C-D). Thus, these findings and independent of apoptosis (Supplementary Figure indicate that the LSH-mediated inhibition of S5B); then we observed that LSH overexpression erastin-induced ferroptosis in cancer cells is decreased the erastin-induced growth inhibition of dependent on iron and lipid ROS. H358 cells (Figure 3A) and PC9 cells (Figure 3B). The

Figure 3. LSH inhibits ferroptosis. (A,B) Response of H358 (A) and PC9 (B) cells with or without LSH overexpression to erastin (5μM) ± Ferrostatin(1μM). (C) Response of A549 cells to erastin (5μM) ± Ferrostatin(1μM) after LSH depletion. Viability was assessed by Alamar blue and represents the mean ± SD from three independent biological replicate experiments. (D-G) The total iron levels (D, F) and ferrous iron level (E, G) were analyzed in H358 cells and PC9 cells with or without LSH overexpression (D, E) and A549 cells with or without LSH depletion (F, G). (H-J) Lipid ROS was measured by C11-BODIPY staining coupled to flow cytometry in erastin-treated H358 (H) and PC9 (I) cells, and A549 cells after the LSH depletion. * P<0.05, ** P<0.01, *** P<0.001.

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Figure 4. Retinoic acid promotes ferroptosis by attenuating LSH expression. (A, B) western blot analysis of LSH in A549 (A) and H522 (B) cells after the treatment of indicated chemicals. (C-E) Volume (C), representative images (D) and weight (E) of A549 tumors in nude mice after treatment with ATRA. (F, G) RT-qPCR analysis of ferroptosis-associated genes in ATRA-treated A549 cells in culture (F) or in ATRA-treated xenograft tumor samples (G). (H) ChIP analysis of WDR76 at the promoters of GLUT1, SCD1 and FADS2 in ATRA-treated A549 cells. (I-K) The total iron (I) and ferrous iron (J) and lips ROS (K) levels in ATRA-treated A549 cells. * P<0.05, ** P<0.01, *** P<0.001.

indicating that the regulation of LSH is related to the Retinoic acid promotes ferroptosis through cellular effects of ATRA (Supplementary Figure S6C). the attenuation of LSH expression Moreover, we found that the iron level and lipid ROS Retinoic acid (ATRA), a metabolite of Vitamin A, levels increased in the A549 cells after ATRA has a critical function in initiating the lineage treatment (Figure 4I-K). These results are consistent differentiation of embryonic stem cells (ESCs), where with the model that ATRA suppresses tumor growth LSH plays a critical role in the maintenance of the by decreasing LSH expression and promoting stem properties [35, 36]. After treating the A549 and ferroptosis. H522 cells with various chemicals for 72 h, we found that ATRA, but not or aspirin, decreased Depletion of the SCD1 and FADS2 metabolic the expression of LSH (Figure 4A-B). Both vitamin E genes induces ferroptosis and erastin did not change LSH expression To determine the physiological roles of the LSH (Supplementary Figure S6A-B). Furthermore, we target genes (i.e., SCD1 and FADS2) in lung found that ATRA reduced A549 tumor volumes carcinogenesis and erastin-induced cell death, we (Figure 4C), tumor formation (Figure 4D) and tumor stably knocked down SCD1 and FADS2 in A549 weights (Figure 4E) at the indicated times in the A549 cancer cells. The knockdown approach successfully cells. To address the potential roles of ATRA in the reduced the SCD1 and FADS2 mRNA levels to less xenograft tumors, we assessed the expression levels of than 15% and 20% respectively (Figure 5A-B). The metabolism-associated genes, including SCD1 and ratio of cell growth was significantly reduced after the FADS2. We found that most of these genes decreased SCD1 (Figure 5C) and FADS2 (Figure 5D) depletion. after ATRA treatment (Figure 4F). Similar Furthermore, the iron levels and lipid ROS increased observations were made in the cultured A549 cells when SCD1 and FADS2, respectively, were depleted after ATRA treatment (Figure 4G). The enrichment of (Figure 5E-J). We also found that erastin-induced cell WDR76 to the indicated genes decreased with the death increased after the knockdown of SCD1 and treatment of ATRA in the A549 cells whereas LSH FADS2 (Figure 5K-L). Moreover, knockdown of SCD1 expression decreased (Figure 4H). A549 cells were and FADS2 decreased the ferroptosis-associated forced expression of LSH after the treatment of ATRA, regulators, such as SOD2, SLC2A4, SLC2A14, then cell viability decreased in response to erastin, SLC7A11, SLC1A5, SLC3A2, CARS, EMC2, RPL8 and

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GLUD1, at the mRNA level (Supplementary Figure all lung cancers (Figure 6F), further indicating that a S7A-B). In summary, these findings suggest that the strong correlation between LSH and EGLN1 exists. LSH target genes SCD1 and FADS2 contribute to Furthermore, we found that the protein expression of ferroptosis. EGLN1 was strongly associated with the expression of LSH in a panel of lung cell lines (Figure 6G). EGLN1 induces LSH expression by repressing To further confirm the link between EGLN1 and HIF1α LSH, we analyzed the EGLN1 protein level after LSH The iron- and oxygen- dependent, was overexpressed (Supplementary Figure S9A) or 2-oxoglutarate-dependent dioxygenase family knocked down in lung cancer cells (Supplementary includes EGLN-1, -2 and -3[37]. To identify the Figure S9B), we did not find that LSH altered EGLN1 potential link between LSH and EGLN1, we analyzed expression. However, EGLN1 was overexpressed in the mRNA levels of EGLNs and LSH in an the cells, and we found that the expression of LSH independent panel of 47 primary lung tumors. was increased (Figure 6H). The knockdown of EGLN1 Compared to the adjacent normal tissues, we found decreased the mRNA expression of LSH and cell that the LSH, EGLN1 and EGLN3 mRNA levels were growth (Figure 6I-J), indicating EGLN1 regulates LSH highly expressed in the lung cancer tumors (Figure expression. We treated the A549 cells with the 6A-C), while the mRNA levels of EGLN2 were following EGLN/PHD inhibitors: unchanged (Data not shown). Furthermore, LSH dimethyloxalyglycine (DMOG), CoCl2, inhibitors of expression was higher in both lung ADC dioxygenases, and BAY 85-3494 (BAY), a specific (Supplementary Figure S8A) and SCC inhibitor of EGLNs with highest potency against (Supplementary Figure S8B). The correlation between EGLN1. We found that inhibition of EGLNs increased LSH and EGLN1 or EGLN3 was further analyzed, and HIF1α and decreased LSH expression at the protein we found that there was a strong correlation between levels (Figure 6K-M), whereas inhibiting of LSH and the EGLNs (Figure 6D-E). Moreover, we EGLN/PHD further decreased ferroptosis in response detected the LSH, EGLN1 and EGLN3 protein levels to erastin (Supplementary Figure S9C). In summary, in four lung cancer cases (case 1: lung ADC, case 2: these data indicate that HIF-1α might function as a lung adenosquamous cancer, and case 3 and 4 : lung repressor of LSH expression. SCC); notably, EGLN1, EGLN3 and LSH increased in

Figure 5. Inhibition of the SCD1 and FADS2 metabolic genes promotes ferroptosis. (A, B) RT-qPCR analysis was conducted to detect SCD1 (A) and FADS2 (B) in A549 cells that were stably transfected with two distinct target gene shRNA expression vectors and in control cells (siCTRL). (C, D) The MTT assay was performed to assess A549 cell viability after stable depletions of SCD1 (C) or FADS2 (D). (E-H) The Total iron and ferrous iron levels were analyzed in A549 cells in the depletion of SCD1 (E, F) and FADS2 (G, H). (I-L) Lipid ROS was measured by C11-BODIPY staining coupled to flow cytometry in A549 cells after the depletion of SCD1 (I) and FADS2 (J). A549 cells responses to erastin (5μM) ± Ferrostatin(1μM) after depletion of SCD1 (K) and FADS2 (L). * P<0.05, ** P<0.01, *** P<0.001.

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Figure 6. The EGLN1/c-Myc axis activates LSH expression. (A-E) RT-qPCR analysis of LSH (A), EGLN1 (B) and EGLN3 (C) expression in 47 lung cancer samples paired with corresponding normal lung tissues. The correlations between LSH and EGLN1 (D), EGLN3 (E) were analyzed. (F) (F, G) Western blot analysis of LSH, EGLN1 and EGLN3 in lung cancer tissues and normal lung tissues (F) and a panel of lung cancer cells (G). (H) Western blot analysis of LSH, EGLN1 and EGLN3 in H358 cells overexpressing LSH or EGLN1. (I) Determination of LSH mRNA level by qRT-PCR after depletion of EGLN1 in A549 cells. (J) MTT assays to assess cell viability in A549 cells after depletion of EGLN1. (K-M) Western blot analysis of the indicated proteins after the treatment with DMOG (K), BAY 85-394 (L) and CoCL2 (M). (N) Western blot analysis of the indicated proteins after ATRA treatment. * P<0.05, ** P<0.01, *** P<0.001.

When HIF-1α is induced, it immediately disrupts vitro (Figure 7A, B), and increased migration and c-Myc complexes [38-40], indicating that inhibition of invasion in an in vitro assay (supplementary Figure HIF-1α and EGLN1 might induce the expression of S11A). To address whether LSH plays a role in lung c-Myc. As expected, we found that the expression of cancer in vivo, we utilized a xenograft model to assess c-Myc decreased after both the inhibition of the tumor formation in nude mice. We found that the EGLNs and the induction of HIF-1α (Figure 6K-M). injection of H358-LSH cells (2×106) significantly Moreover, we showed that EGLN and c-Myc were increased the tumor size (supplementary Figure recruited to the promoter of LSH at two HIF-1α S11B), tumor volume (Figure 7C) and tumor weight binding sites in the lung cancer cells (Supplementary (Figure 7D), while the whole-body weight remained Figure S10A-B), whereas inhibition of EGLN activity unchanged (supplementary Figure S11C). The after the treatment of BAY decreased the binding of expression levels of metabolism-associated genes, EGLN1 and c-Myc to the LSH promoter region including SCD1 and FADS2 were increased by LSH in (Supplementary Figure S10C-D). After the treatment the xenograft tumors (Figure 7E). of CoCl2 in A549 cells, both EGLN1 and c-Myc was Conversely, LSH knockdown resulted in recruited to the LSH promoter region (Supplementary significantly reduced cell growth rate (Figure 7F) and Figure S10E-F) Lastly, ATRA decreased LSH and impaired the formation of colonies (Figure 7G and c-Myc protein expression levels in A549 cells (Figure supplementary Figure 11D). When we injected 3 × 106 6N), which further support the hypothesis that the A549 cells into nude mice, we observed that LSH c-Myc/EGLN1 axis upregulated the expression of depletion significantly reduced the tumor volume LSH expression by recruiting c-Myc to the LSH (Figure 7H), tumor formation and tumor weight promoter. (supplementary Figure S11E and Figure 7I), while the body weight did not significantly change in either LSH functions as an oncogene in lung cancer groups (supplementary Figure S11F). The expression To determine the physiological role of LSH in levels of metabolism-associated genes, including lung cancer, we analyzed the role of LSH in cancer in SCD1 and FADS2 were decreased after depletion of vitro and in vivo. The overexpression of LSH LSH in the xenograft tumors (Figure 7J). significantly increased the growth of all cell lines in

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Figure 7. LSH functions in vitro and in vivo as an oncogene in lung cancer. (A) The MTT assay was used to assess the cell viability of PC9 lung cancer cells that were stably transfected with an LSH expression vector. (B) Growth in soft agar was measured for PC9 cells that stably overexpressed LSH with quantitative analysis. (C) Tumor volumes in nude mice are shown after injection of PC9 cells stably expressing control vector or LSH expression plasmids as indicated time. (D) Tumor weights were recorded. (E) mRNA expression of the indicated genes measured by qPCR in xenograft tumors derived from PC9 cells with highly expression of LSH. (F) The MTT assay was performed to assess the cell viability of A549 cells that were stably transfected with two distinct LSH shRNA expression vectors (siLSH#1 and siLSH#2) and of control cells (Mock). (G) Growth in soft agar was measured for A549 cells that LSH was stably knocked down with quantitative analysis. (H) Tumor volumes in nude mice are shown after injection of A549 cells stably depletion of LSH as indicated time and (I) Tumor weights were recorded. (J) mRNA expression of the indicated genes measured by qPCR in xenograft tumors derived from A549 cells in the depletion of LSH. (K) The LSH protein levels in lung cancer patients were determined by immunohistochemistry. (L) The lung cancer tissue-associated LSH expression levels are significantly elevated. Kaplan-Meier curves for the overall survival rates that are associated with samples measured here for lung cancer (M) and lung ADCs (N). * P<0.05, ** P<0.01, *** P<0.001.

We observed the increase of LSH mRNA GLUT1, SCD1 and FADS2. These metabolic genes increased in lung tumors (Fig 6A) and the increase of inhibit the accumulation of lipid ROS and LSH protein in limited lung tumor samples (Fig 6F). intracellular iron, which are required for ferroptosis, To further determine the role of LSH in lung cancer, and inhibition of ferroptosis by LSH ultimately we performed immunohistochemical analysis on promotes cancer progression. tissues from lung cancer patients. The LSH protein was present in the nuclei of the normal lung tissues, Discussion and its expression was greatly increased in the lung Our findings provide evidence that LSH plays an ADC and SCC tissues (Figure 7K-L). However, we did important role in lung carcinogenesis. Our findings not detect any point mutations in LSH by sequencing are the first to suggest that LSH acts as an important in the 10 lung cancer tissues (data not shown). inhibitor of ferroptosis in carcinogenesis by Notably, a Kaplan-Meier analysis was performed on a promoting lipid metabolism-related genes, which act cohort of patients with these lung cancers and showed as novel key regulators of ferroptosis; moreover, LSH that lower LSH expression was associated with the is upregulated by the EGLN1/c-Myc axis, whereas overall survival in all lung cancers (Figure 7M), and HIF-1α functions as a repressor of LSH expression. ADCs (Figure 7N), but not in lung SCCs A large-scale genome-wide analysis of various (supplementary Figure S11G). Taken together, LSH cancer model systems has revealed a strong functioned as an oncogene in lung cancer progression. correlation between aberrant epigenetic modifications Based on our observations, we propose a model and tumor progression, thereby highlighting the for LSH-mediated inhibition of ferroptosis and importance of chromatin-modifying enzymes in enhancement of lung tumorigenesis (supplementary cancer [41, 42]. Interestingly, chromatin-modifying Figure S12). In this model, LSH acts as a novel enzymes mediate sensing of the intermediary inhibitor of ferroptosis by regulating several metabolism products to modulate gene regulation metabolism-related genes. LSH is upregulated by and disease progression [43]. LSH is critical for c-Myc, which is enriched at the LSH promoter by the chromatin function and the establishment of DNA EGLN1-mediated repression of HIF-1α. The induced methylation [20, 21, 32, 35]. Several reports have LSH interacts with WDR76, which, in turn, shown that LSH contributes to the malignant upregulates the lipid metabolic genes, including progression of prostate cancer, melanoma, head and

http://www.thno.org Theranostics 2017, Vol. 7, Issue 13 3303 neck cancer, nasopharyngeal carcinoma and glioma HIF-2α degradation. HIF-1α regulates [44-47], the mechanism for this remains less known. oxygen-dependent glucose and glutamine We recently indicate that LSH is shown to co-operate metabolism, playing a critical role in cancer with partners, such as G9a to drive cancer progression progression [12, 13, 56, 57]. In fact, EGLN1 inhibition [33, 44, 48]. However, the molecular mechanisms, causes accumulation of circulating metabolites [58]. particularly in lung cancer, are not well understood. Interestingly, some oncometabolites stimulate EGLN Here, we demonstrate that LSH contributes to lung activity, which leads to diminished HIF levels [14]. cancer progression by directly upregulating metabolic For example, high extracellular glutamate levels genes including SCD1 and FADS2. LSH-mediated inhibit the xCT glutamate-cysteine antiporter and increases in metabolic gene expression may occur thereby interfere with cysteine uptake, which results through a DNA methylation-independent mechanism in lower intracellular cysteine levels. Decreased rather than through chromatin regulation [33, 49, 50]. intracellular cysteine levels inhibit EGLN activity and Here, we provide evidence for an interaction between stabilize HIF-1α [59]. We have recently shown that LSH and WDR76, which is a nuclear WD40 protein of oncometabolites also activate LSH expression [33]. unknown function in mammals. The LSH-dependent Here, we have demonstrated that EGLN1 upregulates recruitment of WDR76 to the metabolic gene LSH expression by inhibiting HIF‐1α, which promoters and the subsequent chromatin highlights HIF‐1α as a key repressor of LSH modification that leads to metabolic gene activation, expression. EGLN2 is to be essential for cell death and links epigenetic regulation by LSH to upregulation of is a candidate driver of iron chelation-mediated the emerging metabolic genes. inhibition of cell death [9]. Interestingly, HIF-1α and The ferroptotic mode of programmed necrosis c-Myc counteract each other [38-40]. We found that was recently discovered as an apoptosis-independent c-Myc is recruited to the HIF-1α on the form of cell death in Ras-transformed cells [51]; the LSH promoter in the normoxic state. Lipid ROS K-ras mutant is common in lung cancer. Ferroptotic accumulation is a key characteristic of ferrotosis; we death is morphologically, biochemically, and have shown that several lipid metabolism genes genetically distinct from apoptosis, necrosis (various influence ferroptosis by affecting the lipid ROS and forms), and autophagy. This process is characterized iron levels. by an overwhelming, iron-dependent accumulation of In summary, we demonstrated that LSH is lethal lipid ROS [7, 8]. Here we demonstrate that LSH involved in ferroptosis and is a potential therapeutic decreases the lipid ROS and iron concentrations, target in cancer because of its crucial role in which supports an inhibitory role for LSH in ferroptosis. Previous studies have linked ferroptosis ferroptosis. Ferroptosis is characterized by increased with oncogenic Ras [4], and more recently, the tumor levels of lipid peroxidation, which can be caused by suppressor p53 was demonstrated to positively compound-mediated inhibition of GPX4, or by regulate ferroptosis by transcriptionally inhibiting the indirectly targeting GPX4 through glutathione expression of the cysteine/glutamate antiporter depletion [5]. Erastin, a ferroptosis-inducer, inhibits SLC7A11 [4, 6]; our findings demonstrate that LSH ferroptosis by inhibiting the cysteine/glut